Xi Hualong, Zhang Kaixin, Yin Yanchun, Gu Tiejun, Sun Qing, Shi Linqing, Zhang Renxia, Jiang Chunlai, Kong Wei, Wu Yongge
National Engineering Laboratory for AIDS Vaccine, Jilin University, Changchun 130012, P.R. China.
Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun 130012, P.R. China.
J Microbiol Biotechnol. 2017 Apr 28;27(4):718-724. doi: 10.4014/jmb.1611.11062.
The combination of rabies immunoglobulin (RIG) with a vaccine is currently effective against rabies infections, but improvements are needed. Genetic engineering antibody technology is an attractive approach for developing novel antibodies to replace RIG. In our previous study, a single-chain variable fragment, scFv57R, against rabies virus glycoprotein was constructed. However, its inherent weak stability and short half-life compared with the parent RIG may limit its diagnostic and therapeutic application. Therefore, an acidic tail of synuclein (ATS) derived from the C-terminal acidic tail of human alpha-synuclein protein was fused to the C-terminus of scFv57R in order to help it resist adverse stress and improve the stability and halflife. The tail showed no apparent effect on the preparation procedure and affinity of the protein, nor did it change the neutralizing potency in vitro. In the ELISA test of molecular stability, the ATS fusion form of the protein, scFv57R-ATS, showed an increase in thermal stability and longer half-life in serum than scFv57R. The protection against fatal rabies virus challenge improved after fusing the tail to the scFv, which may be attributed to the improved stability. Thus, the ATS fusion approach presented here is easily implemented and can be used as a new strategy to improve the stability and half-life of engineered antibody proteins for practical applications.
狂犬病免疫球蛋白（RIG）与疫苗联合使用目前对狂犬病感染有效，但仍需改进。基因工程抗体技术是开发新型抗体以替代RIG的一种有吸引力的方法。在我们之前的研究中，构建了一种针对狂犬病病毒糖蛋白的单链可变片段scFv57R。然而，与亲本RIG相比，其固有的稳定性差和半衰期短可能会限制其诊断和治疗应用。因此，将源自人α-突触核蛋白C末端酸性尾巴的突触核蛋白酸性尾巴（ATS）融合到scFv57R的C末端，以帮助其抵抗不利应激并提高稳定性和半衰期。该尾巴对蛋白质的制备过程和亲和力没有明显影响，在体外也没有改变中和效力。在分子稳定性的ELISA试验中，蛋白质的ATS融合形式scFv57R-ATS在血清中的热稳定性增加且半衰期比scFv57R更长。将尾巴融合到scFv后，对致命狂犬病病毒攻击的保护作用得到改善，这可能归因于稳定性的提高。因此，本文提出的ATS融合方法易于实施，可作为一种新策略来提高工程抗体蛋白的稳定性和半衰期以用于实际应用。
